Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24 April 2017 to 8 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Deviation 1
A third correction group was required to be added to the study design due to the potential interference with the MTT reduction assay by the test item. Following an inconclusive result during the pre-test for the direct reduction of MTT, a further correction group (in addition to the water-killed tissues for correcting direct MTT reduction and viable tissues for correcting color interference) was necessary in order to avoid a double correction.
Assessment of Color Interference in non-viable tissues:
For each exposure period, the additional group was composed of two test item treated freeze killed tissues that underwent the entire testing procedure with the exception that the MTT incubation period was replaced by incubation with assay medium (therefore without MTT). For each exposure period, two freeze killed tissues were treated with sterile water for negative control purposes. The optical density measurements from these tissues were then assessed for possible quantitative correction of the results.
Deviation 2
An assessment of the test item’s capability to directly reduce MTT was inconclusive due to the intrinsic color of the test item and therefore, an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred.

Deviation 3
The solution containing the test item was a yellow to green color, therefore additional color correction tissues were incorporated into the testing procedure. However, the results obtained showed that no color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes. As the results of the color correction tissues were not used it was therefore unnecessary to use the results of the killed color correction tissues, as no double correction for color interference would have occurred.
Deviations:
yes
Remarks:
See remarks above
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch 7215607388
- Expiration date of the lot/batch: 04 August 2017
- Purity test date: 15.12.2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was used as supplied

FORM AS APPLIED IN THE TEST (if different from that of starting material)
The test item was used as supplied

In vitro test system

Test system:
artificial membrane barrier model
Source species:
human
Cell type:
other: Reconstructed Human Epithelium
Justification for test system used:
This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM
- Tissue batch number(s): 25817
- Production date: not specified
- Shipping date: not specifed
- Delivery date: 06 June 2017
- Date of initiation of testing: 24 April 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Rinsing was performed at the end of the 3 minutes and at the 60 minutes exposure periods.
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth:not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Not specified

NUMBER OF REPLICATE TISSUES: duplicates were used

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): Freeze-killed tissues were prepared by placing untreated EPIDERMTM tissues in an empty 12 well plate and storing in a freezer (-14 to -30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
- N. of replicates : Two freeze-killed tissues were used
- Method of calculation used: relative viability (%) = (Mean OD570 of test item / Mean OD570 of negative control) x 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 Independant test sequencies were performed : Pre test and Main test

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% and or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
- Concentration (if solution): pure

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): pure

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8.0N
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours (MTT incubation)
Number of replicates:
Duplicates were used

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main Test - Negative Control at 3 and 60 minutes of exposure
Value:
100
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - Positive Control at 3 and 60 minutes of exposure
Value:
3.9
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - Test Item 3 minutes of exposure
Value:
105.8
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - Test Item 3 minutes of exposure
Value:
97.9
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not specifed
- Direct-MTT reduction: An assessment of the test item’s capability to directly reduce MTT was inconclusive due to the intrinsic color of the test item and therefore, an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred.
- Colour interference with MTT: The solution containing the test item was a yellow to green color, therefore additional color correction tissues were incorporated into the testing procedure. However, the results obtained showed that no color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive control ( 8.0N Potassium Hydrxide) induced positive response to the test system

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.556 for the 3 Minute exposure period and 1.507 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control:The relative mean tissue viability for the positive control treated tissues was 3.9% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements:In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table1 :Mean OD570Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue

Exposure Period

MeanOD570of individual tissues

Mean OD570of duplicate tissues

Standard Deviation

Coefficient ofVariation
(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.511

1.556

0.063

4.0

100*

1.600

60 Minutes

1.488

1.507

0.026

1.7

1.525

Positive Control

3 Minutes

0.062

0.061

0.002

na

3.9

0.059

60 Minutes

0.066

0.060

0.009

na

3.9

0.053

Test Item

3 Minutes

1.636

1.646

0.014

0.9

105.8

1.656

60 Minutes

1.380

1.475

0.134

9.1

97.9

1.570

 

 


OD = Optical density

*=          The mean percentage viability of the negative control tissue is set at 100%

na =         Not applicable

Applicant's summary and conclusion

Interpretation of results:
other: not classified as corrosive substance according CLP criteria.
Conclusions:
Under the experimental condition of this study, the registered substance 4-amino-6-chloro-4-nitrophenol did not led to corrosion when applied on EpiDermTM Reconstructed Human Epithelium. Hence, the B099 was not classified as corrosive substance according CLP criteria.
Executive summary:

The purpose of this GLP compliant study is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes according the OECD 431 guideline method.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes.  Negative and positive control groups were treated for each exposure period.  The test item was found to have the potential to cause color interference and therefore additional tissues were incorporated for color correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT loading.  After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

The relative viability after the 3 and 60 minutes exposure periods were respectively 105.8% and 97.9%. The positive and negative control values were withinthe acceptance criterias.

Under the experimental condition of this study, the registered substance 4-amino-6-chloro-4-nitrophenol did not led to corrosion when applied on EpiDermTM Reconstructed Human Epithelium. Hence, the B099 was not classified as corrosive substance according CLP criteria.